QIAGEN LongRange PCR Kit provides a dedicated and highly reliable solution for long-range amplification from genomic DNA and cDNA — even with very low amounts of template. Low error rates are assured using a novel high-fidelity enzyme mix. The kit includes nucleotides and is highly suitable for use in complex genotyping experiments.

The QIAGEN LongRange PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

High yields of very long PCR products.

Long PCR products (27 kb and 24 kb) were amplified from human genomic DNA using the QIAGEN LongRange PCR Kit and 2 additional long-range PCR kits from the indicated suppliers, according to the manufacturer's instructions. The QIAGEN LongRange PCR Kit provided much higher yields of specific product than the other kits that were tested.

Protection of DNA from excessive damage.

Genomic DNA from leukocytes was pretreated in water (Water), long-range PCR buffer from Supplier A (Standard buffer), or QIAGEN LongRange PCR Buffer (LongRange Buffer), and then directly used for PCR (–) or incubated at 95°C (+) before PCR. Amplification reactions (50 μl) were performed using the QIAGEN LongRange PCR Kit. M: marker. Pretreatment of DNA in QIAGEN LongRange PCR Buffer provided protection from damage, resulting in increased yield. Reduced product yields were achieved due to greater DNA damage when DNA was pretreated in water or standard long-range PCR buffers.

Performance

The QIAGEN LongRange PCR Kit contains all of the components required for the amplification of long PCR fragments (up to 40 kb in size). A unique blend of thermostable DNA polymerases, with enhanced processivity, allows the amplification of full-length PCR products — even from very small amounts of template. The enzyme mix provides high extension rates and proofreading ability for increased fidelity (up to 5-fold higher fidelity than Taq DNA polymerase). The novel buffer system provides highly specific long-range PCR amplification, even from complex genomic DNA (see figure "High yields of very long PCR products"). In addition, the buffer affords significant protection against depurination, ensuring full-length amplification (see figure "Protection of DNA from excessive damage"), outperforming products from other suppliers. DNA pre-incubated in LongRange PCR Buffer shows similar PCR product yields compared to undamaged control DNA, demonstrating that the protective function of the buffer system provides an optimal reaction environment for the amplification of long PCR products. Suboptimal PCR can be improved with Q-Solution — an additive for the amplification of GC-rich templates — also provided with the kit.

The QIAGEN LongRange PCR Kit combines a powerful polymerase blend with an innovative buffer system designed for efficient amplification of long targets up to 40 kb. The kit also includes Q-Solution, a PCR additive that facilitates amplification of GC-rich templates, as well as dNTPs.

LongRange PCR Enzyme Mix

The LongRange PCR Enzyme Mix is a mixture of highly pure recombinant thermostable DNA polymerases and processivity-enhancing factors, cloned in E. coli. This special enzyme blend ensures a very high extension rate and increased fidelity, enabling successful amplification of very long fragments.

Optimized buffer system for extremely long PCR products

Amplification of PCR products longer than 4 kb often fails and generally requires lengthy optimization procedures. Reasons for failure include nonspecific primer annealing or suboptimal cycling conditions. Preventing DNA damage, such as DNA depurination, is of particular importance for when amplifying long PCR products, as a single DNA lesion within the template is sufficient to stall the polymerase enzyme. DNA damage during PCR cycling can be minimized with specific buffers that stabilize the pH of the reaction. The QIAGEN LongRange PCR Buffer is optimized for the amplification of PCR products up to 40 kb in size and provides significant protection against depurination (see figure "Protection of DNA from excessive damage").

Amplification of rare and difficult templates

The unique zwitterionic formulation of LongRange PCR Buffer improves pH buffering at high temperatures (72–94°C), minimizing pH-driven template degradation. In combination with Q-Solution (provided), it further facilitates amplification of GC-rich and other “difficult” templates.

Procedure

The QIAGEN LongRange PCR Kit includes a preoptimized protocol for reliable and accurate long-range PCR of fragments up to 40 kb in size. The kit can be successfully used for the amplification of long targets (all targets in the range 0.1–10 kb), very long targets (genomic DNA >10 kb; episomal DNA 10–40 kb), long cDNA templates from reverse transcription reaction, as well as GC-rich and other "difficult" templates.

Applications

The QIAGEN LongRange PCR provides accurate long-range amplification of standard and complex templates (even low-abundance or GC-rich templates) and is highly suitable for all PCR applications, including sequencing or targeted resequencing, validation of copy number variants (CNVs), mapping of chromosomal translocation breakpoints and other structural variations, as well as cloning.